Adenosine modulates many important physiological processes by triggering ubiquitously expressed adenosine receptors. This project is focused on ecto-5'-nucleotidase (CD73), a glycosyl phosphatidylinositol (GPI)-linked membrane protein that catalyzes the extracellular dephosphorylation of AMP to adenosine. Until recently, it has been difficult to evaluate the contribution of CD73 to adenosine receptor signaling, as there are multiple routes of adenosine formation in vivo. During the last funding period, experiments with our cd73-/- mice made it clear that CD73-generated adenosine plays a major role in regulating inflammatory responses in many experimental settings and especially in controlling leukocyte migration across different types of endothelial barriers. For example, cd73-/- mice show increased vascular leak and tissue neutrophil accumulation under conditions of hypoxia. The vascular leak is caused by a lack of A2B adenosine receptor (A2BAR) signaling on non-hematopoietic cells while lack of A2BAR signaling on neutrophils contributes to increased neutrophil migration into tissues. Exciting new data suggest that CD73-generated adenosine also regulates the expression of thrombomodulin, an important anticoagulant and anti-inflammatory protein. We hypothesize that the response of cd73-/- mice to hypoxia may be caused, at least in part, by inadequate thrombomodulin levels. Cd73-/- mice also show increased lymphocyte migration across high endothelial venules (HEV) of draining lymph nodes. This is also due to lack of A2BAR signaling in non-hematopoietic cells. In the next funding period, we will investigate the mechanisms by which CD73-generated adenosine regulates leukocyte migration across these two distinct types of endothelial barriers with different degrees of permeability and unique properties including characteristic junction associated proteins, adhesion molecules, and/or signal transduction machinery (Aims I and II). We will also use multiple strategies to determine whether the GPI anchor of CD73 is needed for the efficient coupling of adenosine generation to adenosine receptor signaling. The Specific Aims are: Aim I. To determine the mechanism by which CD73-generated adenosine regulates neutrophil migration across vascular endothelial barriers during hypoxia. Aim II. To determine the mechanism by which CD73-generated adenosine regulates lymphocyte migration across HEV into draining lymph nodes. Aim III. To determine whether the GPI anchor of CD73 is necessary for its ability to modulate adenosine receptor signaling. The results of these experiments will give important new insights into the mechanism by which CD73- generated adenosine regulates leukocyte migration during inflammation and may lead to new therapies for treatment of a variety of disorders including sepsis, cardiovascular disease, inflammation, and autoimmunity. PUBLIC HEALTH RELEVANCE: The overall goal of this proposal is to better understand how inflammation and thrombosis are regulated by adenosine. The roles of an enzyme that produces adenosine and various adenosine receptors will be investigated. A previously unappreciated link between adenosine receptor signaling and the control of thrombosis will be explored. The results of the proposed studies may lead to the development of new therapies for a variety of serious medical conditions that affect millions of Americans including sepsis, inflammation, and autoimmunity.